EP1778213B1

EP1778213B1 - A method for the synthesis of oligomeric compounds

Info

Publication number: EP1778213B1
Application number: EP05774962.4A
Authority: EP
Inventors: Richard A. Gross
Original assignee: BTG International Ltd
Current assignee: BTG International Ltd
Priority date: 2004-07-16
Filing date: 2005-07-15
Publication date: 2017-04-05
Anticipated expiration: 2025-07-15
Also published as: EP1778213A2; US20070208081A1; EP1778213A4; WO2006020137A3; US7947736B2; WO2006020137A2

Description

The present invention relates to a method for the synthesis of a compound of the formula H(OCH(CH₃)CH₂C(O))₃-O-A-O-R wherein A is the residue of 1,3-butandiol and R is H, or H(OCH(CH₃)CH₂C(O))₃- that have utility as nutraceuticals and/or as medicaments for producing ketosis in humans and animals for nutraceutical or therapeutic purposes.
It is known that ketone bodies, particularly (R)-3-hydroxybutyrate (D-β-hydroxybutyrate) and acetoacetate, have both nutritional and therapeutic applications in man and many species of animals. US 6,136,862 and US 6,232,345 relate to the use of D-β-hydroxybutyrate, oligomers, esters and salts thereof, inter alia, in the treatment of cerebral edema and cerebral infarction. US 6,207,856 and PCT/US99/21015 also refer to β-hydroxybutyrate and its oligomers, esters and salts thereof in protecting against other forms of neurodegeneration inter alia, through their proposed ability to activate the tricarboxylic acid (TCA) cycle and, through favourable redox reactions in cells and antioxidant activity, scavenge free radicals. β-Hydroxybutyrate has also been demonstrated to have cardioprotectant effect and can increase cardiac efficiency (Sato et al, FASEB J, 9: 651-658, 1995).
It is also known, based on studies of the EEG power spectra measured in freely-moving rats, that D-β-hydroxybutyrate and the induction of ketosis have therapeutic utility in the treatment of conditions including, but not limited to, affective and related psychiatric disorders, which include depression, anxiety, schizo-affective disorder, obsessive-compulsive disorder, panic disorder, social anxiety disorder, generalized anxiety disorder and post-traumatic stress disorder, impaired cognitive function and pain ( WO2005/107875 ; PCT/GB2005/01835 ); also in providing symptomatic relief in the treatment of Parkinson's Disease (PD) and other CNS disorders resulting from dopamine deficiency in the brain and also treating and preventing attention deficit hyperactivity disorder (ADHD) and related CNS disorders of cognition, impulsiveness, attention and aggression in children, adolescents and adults ( WO2006/098767 and WO2006/034361 , corresponding to US Provisional Applications 60/611301 and 601611302 ).
US6,207,856 , US6,136,862 and PCT/US99/21015 , teach that preferred ketogenic precursors for producing such ketosis are monohydric-, dihydric and trihydric alcoholic esters of (R)-3-hydroxybutyrate, but particularly a (R)-3-hydroxybutyryl ester of (R)-1,3-butandiol, more preferably the diester formed from two molecules of (R)-3-hydroxybutyrate and one molecule of (R)-1,3-butandiol.
However, it is also known that production of oligomers of (R)-3-hydroxybutyrate in pure form is problematic. PCT/US99/21015 exemplifies a ketogenic oligomer with a mixture of (R)-3-hydroxybutyrate trimer, tetramer and pentamer and exemplifies esters thereof with acetoacetyl trimer, tetramer and pentamer of (R)-3-hydroxybutyrate. Similarly, Hiraide et al (1999) J. Parenteral and Enteral Nutrition Vol 23. No 6 discloses the use of a mixture of dimer and trimer of (R)-3-hydroxybutyrate for studies performed to determine the ability of plasma to degrade these molecules to the monomer, (R)-3-hydroxybutyrate.
In order to ensure that previously untested material is safe and appropriate for human administration, by any route, it is first necessary to evaluate all of its significant components for toxicity and efficacy. In the case of multi-component materials, it is, therefore, necessary to evaluate each one of the components in a variety of toxicology and efficacy tests. Such tasks can be extremely expensive and time-consuming, and inevitably, this will be an important factor in any decision on whether or not to proceed with any particular assessment. Furthermore, a mixture of different components may need to be produced in a set ratio for its safety and efficacy evaluation to be valid.
The present invention provides a method for the synthesis of various, single component oligomeric ketogenic compounds, which are suitable for use in animals and man for therapeutic and/or nutraceutical purposes.
The present invention provides a method for the synthesis of a compound of general formula H(OCH(CH₃)CH₂C(O))₃-O-A-O-R wherein A is the residue of 1,3-butandiol and R is H, or H(OCH(CH₃)CH₂C(O))₃- comprising reacting a cyclic oligomer of (R)-3-hydroxybutyrate consisting of three (R)-3-hydroxybutyrate moieties with 1,3-butanediol in the presence of Candida antarctica lipase type B in a furan or pyran solvent.
In a preferred embodiment, R is H, the cyclic oligomer is (R)-3-hydroxybutyrate triolide, the alcohol is 1,3-butandiol, the solvent is tetrahydrofuran (THF) and the lipase is Candida antarctica lipase type B (CAL-B), particularly that available from Novozyme as Novozyme 435.
Preferably the method is that wherein the 1,3-butandiol is racemic and in one preferred form of that method the product provided is at least 95% mono-adduct wherein R is H.
A further preferred method is that wherein the solvent is tetrahydrofuran and the lipase is CAL-B. Again, the method is preferably that wherein the enzyme is Novozyme 435.
Still more preferably, the solvent is a mixture of tetrahydrofuran and toluene in ratio in the range 10:90 to 90:10 and the lipase is CAL-B.
For such method the product is a mixture of mono and di-adducts of H(OCH[CH₃]CH₂C[O])₃- with (R)-1,3-butandiol in ratio of 10:90 to 90:10.
In a convenient embodiment of the invention, the products are separated and purified by column chromatography using a methanol:chloroform mixture. More preferably the mixture is between 1 and 5% methanol and 99 to 95% chloroform v/v.
Cyclic esters of (R)-3-hydroxybutyrate are known in the art and are readily produced by known methods: see for example Seebach et al. Helvetia Chimica Acta Vol 71 (1988) pages 155-167, and Seebach et al. Helvetia Chimica Acta, Vol 77 (1994) pages 2007 to 2033.
The method produces esters of (R)-3-hydroxybutyrate having efficacy in raising ketone body levels on adminstration to humans and animals, particularly, but not exclusively, by the oral, parenteral or intravenous route.
The method produces compounds of formula H(OCH(CH₃)CH₂C(O))₃-O-A-O-R wherein A is the residue of 1,3-butandiol and R is H, or H(OCH(CH₃)CH₂C(O))₃-.
One particular group of diesters of (R)-1,3-butandiol which can be prepared with the method of the invention are those wherein R is H(OCH(CH₃)CH₂C(O))₃-. Most conveniently these compounds are diesters of one equivalent of (R)-1,3-butandiol with two equivalents of the trimer of (R)-3-hydroxybutyrate. These compounds have advantage in that all of the component alcohol and acid moieties are converted to energy producing elements in the body on administration.
The method can provide compounds in pure form, preferably containing no more than 10% by weight, more preferably no more than 5% by weight, of other (R)-3-hydroxybutyrate moiety containing compounds.
Particularly the method can provide compounds in at in least 90% weight/weight pure form, more preferably at least 95% w/w form. More particularly at least 98% w/w form
The compounds produced with the method of the present invention can be used to prepare a nutraceutical or pharmaceutical composition together with a foodstuff component or a pharmaceutically acceptable carrier, diluent or excipient. Such foodstuff components may be selected for example from the common food groups such as carbohydrates, proteins, fats and vitamins. Carbohydrates may for example be selected from mono-, oligo- and polysaccharides or may even be alternative mono-, oligo- and polymeric forms of (R)-3-hydroxybutyrate.
The compounds prepared according to the method of the present invention can be used for the manufacture of a medicament for producing a physiologically acceptable ketosis. Such medicament will be suitable for treating a number of debilitating conditions that will benefit from the induction of ketosis. These include, but are not limited to, damage to the major organs such as occurring during hemorrhage, myocardial infarction, congestive heart failure, pulmonary infarction, kidney failure, where the medicament may be administered via the appropriate clinical route, which includes, but is not limited to, oral and intravenous.
Where the medicament is intended to be given intravenously, the compounds may be formulated as solutions. The compounds have advantage that many of them are water soluble and do not require counterions, such as the sodium, potassium or calcium that is conventional with molecules such as the (R)-3-hydroxybutyrate monomer, acetate or lactate.
Particularly suitable carriers, diluents and excipients which may be utilised for eg. oral and parenteral, formulations, may be selected from conventional options such as those disclosed in the patents listed herein, or in Remington, The Science and Practice of Pharmacy 21st Edition, Edit David B Troy ISBN 0-7817-4673-6. for example, see Chapter 39 for Solutions. Emulsions, Suspensions, Syrups, Elixirs and Extracts. For example, Elixirs are clear, optionally flavoured liquids for oral use.
The compounds may also be used for the treatment of neurological and psychiatric conditions, such as stroke, head trauma, neurodegenerative conditions, including Alzheimer's and Parkinson's diseases, epilepsy, pain, spasticity and related motor disorders, affective and related psychiatric disorders, including depression, anxiety, schizo-affective disorder, obsessive-compulsive disorder, panic disorder, social anxiety disorder, generalized anxiety disorder and post-traumatic stress disorder, attention deficit hyperactivity disorder (ADHD) and related CNS disorders of cognition, impulsiveness, attention and aggression, diabetes, and obesity. These compounds can also be used to prevent cytotoxic damage to all end-organ systems, including, but not limited to, the central nervous system including its associated sensory, motor, cognitive and emotional function, heart, lung, liver, kidney and gut, such as occurring during major surgery.
The present invention will now be described further by reference to the following non-limiting Examples, Schemes and Figures. Further embodiments falling within the scope of the claim will occur to those skilled in the art in the light of these.

FIGURES

Figure 1 : shows the reaction scheme for synthesis of the triolide of (R)-2-hydroxybutyrate.
Figure 2 : shows the reaction scheme for synthesis of the mono-adduct (KTX0202).
Figure 3 : shows the reaction scheme for synthesis of the mono-adduct and di-adduct KTX0202 and 0203.
Figure 4 : shows ketogenic effect of oral administration KTX 0101 (sodium β-hydroxybutyrate) as determined by increases of β-hydroxybutyrate concentrations in rat plasma. Animals were dosed with KTX 0101 (300mg/kg po) and killed at the relevant time-points for collection of plasma. Samples were assayed with the standard Ranbut assay using a 20µl sample volume and values are shown as mean ± SEM, n=4. One-way analysis of variance followed by Dunnett's test was used to compare post-dose time-points to the baseline (T=0) control; ***P<0.001, *P<0.05.
Figure 5 : shows ketogenic effect of oral administration of the mono-adduct (KTX 0202) as determined by increases of β-hydroxybutyrate concentrations in rat plasma. Animals were dosed with KTX 0202 (300mg/kg po) and groups (n=4) were killed at 30, 120 and 240 minutes for collection of plasma. Terminal cardiac plasma samples were also taken at baseline (T=0). Samples were assayed using the Ranbut β-hydroxybutyrate spectrophotometric assay with a 20µl sample volume. Values are shown as mean ± SEM, n=4. One-way analysis of variance followed by Dunnett's test was used to compare post-dose time-points to the baseline (T=0) control; ***P<0.001, *P<0.05.
Figure 6 : Ketogenic effect of oral administration of the di-adduct (KTX 0203) as determined by increases of hydroxybutyrate concentrations in rat plasma. Animals were dosed with KTX 0203 (300mg/kg po) and groups (n=4) were killed at 30, 120 and 240 minutes for collection of plasma. Terminal cardiac plasma samples were also taken at baseline (T=0). Samples were assayed using the Ranbut β-hydroxybutyrate spectrophotometric assay with a 20µl sample volume. Values are shown as mean ± SEM, n=4. One-way analysis of variance followed by Dunnett's test was used to compare post-dose time-points to the baseline (T=0) control; ***P<0.001.
Figure 7 : Shows ketogenic effect of oral administration of the (R)-3-β-hydroxybutyrate triolide as determined by increases of plasma hydroxybutyrate concentrations in rat plasma. Animals were dosed and groups (n=6) killed at the indicated time-points for collection of whole blood and subsequent collection of plasma. Samples were assayed using the Ranbut spectrophotometric assay described (20µl sample size) and individual values are the mean of triplicate determinations. Group means are shown as ± SEM, n=6. Statistical comparisons against baseline (t=0) were by multiple t-tests (***p<0.001).
Figure 8 : Shows ketogenic effect of intravenous injection of the mono-adduct (KTX 0202) as determined by increases of plasma hydroxybutyrate concentrations in rat plasma. Animals were dosed with an intravenous bolus and groups (n=6) killed 15 minutes later for the collection of whole blood and subsequent collection of plasma. Samples were assayed with the Ranbut spectrophotometric assays (20µl sample size) and individual values are the mean of triplicate determinations. Group means are shown as ± SEM, n=6. Statistical comparisons against baseline (t=0) were by multiple t-tests (***p<0.001).

EXAMPLES

General procedure for the synthesis of R-3-hydroxybutyrate triolide from PHB [poly(3-hydroxybutyrate)] (Figure 1)

A mixture of poly(3-hydroxybutyrate) (PHB; 36g) and toluene-4-sulphonic acid monohydrate (23g) in 750 ml of toluene/dichloroethane (4:1) was heated and stirred at reflux for 20hrs. The water was removed azeotropically using a Dean-Stark trap for 15 hrs. The resulting clear brown solution was cooled to room temperature, washed with a half-saturated solution of Na₂CO₃, then washed with a saturated solution of NaCl, dried over MgSO₄, filtered and evaporated to dryness. The crude mixture was purified by column chromatography using silica gel as the stationary phase and a hexane/ethylacetate mixture as the eluent followed by repeated crystallizations. The yield of the purified product was 15g (40%).
Analysis of the product was as follows: melting point 110°C; ¹H NMR (300 MHz, CDCl₃) : δ 1.30 (9H, d,J=6.4 Hz, CH₃), 2.39 (3H, dd, J=2.1, 11.3 Hz), 2.46 (3H, dd, J=11.3, 13.5Hz) and 5.31-5.39 (3H, m, CH); ¹³C NMR (75.5 MHz, CDCl₃) : δ 20.76 (CH₃), 42.16 (CH₂), 68.87 (CH) and 170.01 (CO).

LIPASE CATALYZED TRANS-ESTERIFICATION PROCESS:

EXAMPLE 1: Synthesis of 1-(tri(R)-3-hydroxybutyryl-butane-1,3-diol) ester (KTX0202)

The enzyme-catalyzed ring-opening of the (R)-3-hydroxybutyrate triolide (cyclic trimer ester) with racemic 1,3-butandiol was studied as a function of the lipase source, reaction temperature, solvent, and time. The results of this work are given in Table 1.

Into a round-bottom flask (50 ml) was added purified triolide (100 mg, 0.387 mmol), (R,S)-1,3-butandiol (69.8 mg, 0.774 mmol), 1 ml of anhydrous organic solvent, and lipase (34 mg, 20%-by-wt relative to total substrates). The reactions were performed at selected reaction temperatures and times (Table 1). The reactions were terminated by first cooling the reaction mixture to room temperature, removing the enzyme by filtration, and then stripping the solvent by rotary-evaporation. A total of 525 reactions were carried out.

TABLE 1:

Enzyme	Solvent (1)	Temperature (°C)	Time (hr)	Result
Candida cylindrecea lipase (CCL)	1 to 5	1 to 5	1 to 3	Negative
Porcine pancreatic lipase (PPL)	1 to 5	1 to 5	1 to 3	Negative
Amano pseudomonas cepecia (PS)	1 to 5	1 to 5	1 to 3	Negative
Candida antarctica lipase (CAL-B)	1 to 5	1 to 5	1 to 3	Positive in one solvent
Mucor meihei
	1 to 5	1 to 5	1 to 3	Negative
Lipase pseudomonas fluorescence (PF)	1 to 5	1 to 5	1 to 3	Negative

Solvents : (1) tetrahydrofuran (THF) (2) toluene, (3) acetonitrile, (4) dioxane and (5) diethyl ether. Temperatures : (1) 45, (2) 50, (3) 55, (4) 60 and (4) 70°C. Reaction times : (1) 24, (2) 48, (3) 72 hrs.
CAL-B in THF at 55°C was the best system for carrying out this reaction. No other lipase was found to be active. The product was purified by column chromatography using silica gel as the stationary phase and MeOH/CHCl₃, 2% v/v as the eluent. Analysis of the product was as follows:

Yield: 61% (82 mg) of a transparent oil; IR (CHCl₃) cm^-1 : 3447, 2926,1750, 1392 and 1197; ¹H NMR (300 MHz, CDCl₃): δ 1.20 (12H,d,J= 6.4Hz, CH₃), 1.70-1.78 (4H,m,H-3,COCH₂), 2.38-2.46 (4H,m,COCH₂ ×2), 3.11(1H,brs,OH exchangeable with D₂O), 3.88-3.95(1H,m,CH), 4.16-4.21 (3H,m,OCH_a,2XCH), 4.31-4.38 (1H,m,CH_b) and 5.13-5.34(1H,m,CH); ¹³C NMR (CDCl₃): δ 20.81,22.92 (CH₃), 38.17(C-3),43.68(COCH₂×3), 62.48(C-4), 64.66(C- 2), 65.49(CH) and 173.41 (CO); MS (FAB, 3-NBA) [M] ⁺ (% int): 349.23[(M+H)⁺,13], 307.13(26), 289.11(18), 263.17(18), 219.20(8), 177.13(48), 154.06(100) and 137.05(77).

Thus, this is consistent with the mono-(tri-(R)-3-hydroxybutyrate) adduct, being metabolically equivalent to four units of (R)-3-hydroxybutyrate. In the examples below the mono-adduct is referred to as tetramer, containing three (R)-3-hydroxybutyrate moieties and one butandiol-providing four metabolic equivalents of (R)-3-hydroxybutyrate on metabolism. Similarly the di-adduct is referred to as the heptamer, having six (R)-3-hydroxybutyrate moieties and one butandiol.

EXAMPLE 2: Synthesis of 1-(tri(R)-3-hydroxybutyryl-butane-1,3-diol (KTX 0202; tetramer) (Figures 2 and 3) and of 1-3 butandiol, di-(tri(R)-3-hydroxybutyryate) ester (KTX 0203; heptamer) (Figure 3)

After successfully completing the ring-opening of triolide using (R,S)-1,3-butandiol, the reaction was repeated as above (CAL-B, in THF, 72 hrs, 55°C) except that (R)-1,3-butandiol was used in place of its racemate. Thus, into a round-bottom flask (50 ml) was added purified triolide (100 mg, 0.387 mmol), (R)-1,3-butandiol (69.8 mg, 0.774 mmol), 1 ml of an anhydrous organic solvent, and CAL-B (34 mg, 20%-by-wt relative to total substrates). Unexpectedly, in addition to the tetramer that was the primary product using racemic butandiol, the corresponding heptamer was found as a co-product. These two products were separated by column chromatography (MeOH/CHCl₃, 2:98 v/v). The structure of tetrameric product was confirmed as follows:

Yield: 42% (56 mg) of a transparent oil; ¹H NMR (300 MHz, CDCl₃): δ 1.20 (12H,4d,J= 6.4Hz, CH₃), 1.70-1.83(4H,m,H-3,COCH₂), 2.41-2.53 (4H,m,COCH₂ ×2), 3.42, 3.69 (2H,brs,OH exchangeable with D₂O), 4.19-4.24(1H,m,CH), 4.29-4.38 (3H,m,OCH_a,2XCH),4.40-4.45(1H,m,CH_b) and 5.12-5.18(1H,m,CH); ¹³C NMR (75 MHz, CDCl₃): δ 22.63,23.55 (CH₃), 37.68(C-3),43.23(C-6,C-10,C-14), 62.11(C-2,C-15), 64.29(C-7,C-11), 64.89(C-4) and 172.91 (CO); CHN Analysis: Anal. Calcd for C₁₆H₂₈O₈: C, 55.16; H, 8.10; 0,36.74.Found: C, 54.03; H, 8.85; O, 37.12.

EXAMPLE 3: Synthesis of 1-(tri(R)-3-hydroxybutyryl-butane-1,3-diol (KTX 0202; tetramer) (Figures 2 and 3) and of 1,3 butandiol, di-(tri(R)-3-hydroxybutyryate) ester (KTX 0203; heptamer) (Figure 3)

The structure of heptamer product was confirmed as follows: Yield: 35% (82mg) of a transparent oil; ¹H NMR (300 MHz, CDCl₃): δ 1.24-1.30 (21H,m, CH₃), 1.87-1.93 (4H,m,H-17,2XCOCH₂), 2.38-2.63 (14H,m,COCH ₂ ×7), 2.96- 3.09(1H,m,OH), 4.11-4.19 (7H,m,5XCH,OCH₂), and 5.09- 5.34(2H,m, 2 XCH); ¹³C NMR (75.0 MHz, CDCl₃): δ 19.82, 22.61,23.49 (CH₃), 37.80,38.80,40.83,43.44, 62.11 (CH₂), 64.40,64.52,67.52,67.88,69.82(CH), and 169.59,170.72,170.47,171.88 (CO). CHN Analysis: Anal. Calcd for C₂₈H₄₆O₁₃: C, 56.94; H, 7.85; O, 35.21. Found: C, 54.26; H, 8.28; O, 37.46.
The ratio of the mono to di-adduct produced in the reaction mixture before separation could be altered by solvent changing. Thus by using a 1:1 mixture v:v of THF to toluene, the ratio of mono:di was changed to 2:3, the reaction being monitored using LC:MS. The formation of the heptamer might occur through the triolide ring opening by action of the tetramer. However, it is believed that repetitive transfer of butyrate units from one tetramer to another is the preferred reaction. Early in the reaction only one peak at 371.27 (M+Na) corresponding to the mono adduct was detected followed by transfer of trimer between molecules to give the di-adduct and some (R)-3-hydroxybutyrate monomer, which was subsequently removed in the purification step.

EXAMPLE 4: Ketogenic activity after oral administration of esters of the invention.

Male Sprague-Dawley rats (weight range 200-250g; Charles River, Margate, Kent) were used. The rats were group-housed in polypropylene cages at a temperature of 21±4°C, 55±20% humidity on a standard 12 hour light/dark cycle (lights on at 6.00am). Animals had free access to a standard pelleted rat diet and tap water at all times. Animals were acclimatized to these conditions for at least one week before experimentation.

The sodium salt of D-β-hydroxybutyrate monomer (KTX 0101) was used as a comparator for assessing the bioavailability of the oligomeric compounds described here. KTX 0101 (Sigma 298360 Lot 18927HB), KTX 0202 or KTX 0203 were administered by oral gavage (po) at 300mg/kg. Control animals received the appropriate vehicle (deionized water at 1 ml/kg) via the same route.

Protocol A: KTX 0101 only

Group	Number of animals	Time of blood sampling (min)	Treatment
3	4	0	Vehicle dosed
4	4	15	KTX 0101 (sodium D-β-hydroxybutyrate)
			300mg/kg po
5	4	30	KTX 0101
			300mg/kg po
6	4	60	KTX 0101
			300mg/kg po

Protocot B: KTX 0202 and KTX 0203 only

Group	Number of animals	Time of blood sampling (min)	Treatment
A
	4	0	Vehicle baseline
B
	4	30	KTX 0202 300mg/kg po or KTX 0203 300mg/kg po
C
	4	120	KTX 0202 300mg/kg po or KTX 0203 300mg/kg po
D
	4	240	KTS 0202 300mg/kg po or KTX 0203 300mg/kg po

Protocol E: (R)-3-β-hydroxybutyrate triolide only

Group	Number of animals	Time of blood sampling (min)	Treatment
A	6	0	Vehicle baseline
B	6	60	(R)-3-β-hydroxybutyrate triolide 300mg/kg po
C	6	120	(R)-3-β-hydroxybutyrate triolide 300mg/kg po

In Protocol A, blood (approx 10 ml) was collected by cardiac puncture at 15, 30 and 60 minutes after dosing. In Protocol B, blood (approx 5 ml) was collected by cardiac puncture at 30, 120 and 240 minutes after dosing. In Protocol E, blood (approx 5 ml) was collected by cardiac puncture at 120 and 240 minutes after dosing. Animals were killed by CO₂ asphyxiation and blood collected by cardiac puncture. Blood was placed into EDTA-coated plasma collection tubes (Sarstedt 5 ml K2E tubes) and kept on ice prior to centrifugation. Tubes were centrifuged in an Eppendorf 570R centrifuge at 4°C for 5 minutes at 2500rpm (1000g). Plasma samples were initially frozen on dry ice and transferred to a -75°C freezer until required for analysis.
Commercial clinical assay kits (Ranbut) for the determination of D-β-hydroxybutyrate were obtained from Randox Laboratories (Antrim, UK). The kit quantified NADH via the activity of β-hydroxybutyrate dehydrogenase measured as an increase in OD340nm. An alkaline pH is necessary to drive the reaction equilibrium towards the production of NADH and acetoacetate.
This spectrophotometric assay was modified for application to a 96-well microplate format. The reaction rate was then determined from the increase in OD340nm over a I minute time-course, after allowing a necessary period for the reaction rate to settle.
KTX 0101 (sodium β-hydroxybutyrate): After oral administration at 300 mg/kg, the sodium salt of the monomer rapidly increased plasma concentrations of β-hydroxybutyrate (Figure 5). The maximum increase was 0.4mM observed at 15 min (P<0.001), it plateaued0 at this level until 30 min (P<0.001) and declined thereafter. The level β-hydroxybutyrate was still significantly (P<0.05) elevated at 1 h, but it returned to control values by 2 h.
KTX 0202 (morto-adduct): After oral administration at 300 mg/kg, the tetramer also significantly increased plasma concentrations of β-hydroxybutyrate (Figure 6). The maximum increase was 0.4 mM observed at 30 min (P<0.001); it declined thereafter, but was still significantly (P<0.05) elevated at 2 h. Plasma β-hydroxybutyrate levels returned to control values by 4 h.
KTX 0203 (di-adduct): After oral administration at 300 mg/kg, the heptamer also significantly increased plasma concentrations of β-hydroxybutyrate (Figure 7). The maximum increase was 0.4mM observed at 30 min (P<0.001); it declined thereafter and plasma β-hydroxybutyrate levels returned to control values by 2 h.
(R)-3-β-hydroxybutyrate triolide: After oral administration at 300 mg/kg, the triolide significantly increased plasma concentrations of β-hydroxybutyrate (Figure 10). The maximum increase was 0.5 mM observed at 60 min (P<0.001); it declined thereafter, but was still significantly (P<0.001) elevated at 2 h.

EXAMPLE 5: Ketogenic activity after intravenous injection of esters of the invention.

Male Sprague-Dawley rats (weight range 200-250g) were obtained from Charles River, Margate, Kent. The rats were group housed in polypropylene cages at a Temperature of 21±4°C, a humidity of 55±20% and on a standard light/dark cycle. Animals had free access to a standard pelleted rat diet and tap water at all times. Animals were accustomed to these conditions for at least one week before experimentation.
KTX 0101 (synthesized by KetoCytonyx) was dissolved at 300mg/ml in deionized water which produced a clear solution of pH 7.0. Similarly, KTX 0202 was dissolved at 300mg/kg in deionized water and produced a slightly opaque solution with a pH of 6.0. The compounds were infused via the tail vein at 300mg/kg (1ml/kg) over a period of 1 to 2 min. Groups for each of the above compounds:

Group Number of animals Time of blood sampling (min) Treatment

A 6 15 Vehicle

B 6 15 KTX 0101

C 6 15 KTX 0202

Animals were killed 15 min later by CO₂ asphyxiation and blood was collected by cardiac puncture. Blood was placed into EDTA-coated plasma collection tubes (Sarstedt 5 ml K2E tubes) and kept on ice prior to centrifugation. Tubes were centrifuged in an Eppendorf 570R centrifuge at 4°C for 5 minutes at 2500rpm (1000g). Plasma samples were initially frozen on dry ice and transferred to a -75°C freezer until required for analysis.
Commercial clinical assay kits (Ranbut) for the determination of D-β-hydroxybutyrate were obtained from Randox Laboratories (Antrim, UK). The kit quantified NADH via the activity of β-hydroxybutyrate dehydrogenase measured as an increase in OD340nm. An alkaline pH is necessary to drive the reaction equilibrium towards the production of NADH and acetoacetate.
KTX 0101 (sodium β-hydroxybutyrate): After intravenous injection at 300 mg/kg, the sodium salt of the monomer rapidly increased plasma concentrations of β-hydroxybutyrate (Figure 11). An increase to 1.0 mM was recorded at 15 min (P<0.001).
KTX 0202 (mono-adduct): After intravenous injection at 300 mg/kg, the tetramer also rapidly increased plasma concentrations of β-hydroxybutyrate (Figure 11). An increase to 1.7 mM was recorded at 15 min (P<0.001).

Discussion

These results demonstrate that KTX 0202 (mono-adduct, tetramer of (R)-3-β-hydroxybutyrate) and KTX 0203 (di-adduct, heptamer of β-hydroxybutyrate) are both orally bioavailable and are capable of producing ketosis when given to rats. The magnitude of the increases in plasma β-hydroxybutyrate evoked by KTX 0202 and KTX 0203 are at least as great as those produced by KTX 0101, the sodium salt of β-hydroxybutyrate. Moreover, in the case of KTX 0202, this β-hydroxybutyrate oligomer has an improved biological half-life relative to KTX 0101.
The (R)-3-β-hydroxybutyrate triolide was also able to increase plasma concentrations of β-hydroxybutyrate after being administered via the oral route, as disclosed in WO00/15216 . Once again, the elevation of plasma β-hydroxybutyrate was at least as great as that evoked by oral administration of the equivalent dose of KTX 0101. Like KTX 0202, (R)-3-β-bydroxybutyrate triolide has an improved biological half-life relative to KTX 0101.
The results also demonstrate that KTX 0202 (mono-adduct, tetramer of (R)-3-β-hydroxybutyrate) is bioavailable when injected via the intravenous route of administration. When injected at identical doses, ie 300 mg/kg iv, KTX 0202 increased the plasma concentration of β-hydroxybutyrate at 15 min by 60% more than KTX 0101.
Together, these results report a route for the synthesis of the novel oligomers of (R)-β-hydroxybutyrate having advantage of being water soluble and defined for purposes of producing ketosis in subjects. The experimental data also demonstrate that these compounds are biovailable when given by both the oral and intravenous routes of administration. Thus, these compounds will have utility as nutraceuticals, or as medicaments where they can be administered using clinically relevant routes, eg orally or intravenously.

Claims

A method for the synthesis of a compound of formula

H(OCH(CH₃)CH₂C(O))₃-O-A-O-R

wherein A is the residue of 1,3-butandiol and R is H or H(OCH(CH₃)CH₂C(O))₃- comprising
reacting a cyclic oligomer of (R)-3-hydroxybutyrate consisting of three (R)-3-hydroxybutyrate moieties with 1,3-butanediol in the presence of Candida antarctica lipase type B in a furan or pyran solvent.
A method as claimed in Claim 1 wherein the solvent further includes toluene.
A method as claimed in Claim 1 wherein the cyclic oligomer is (R)-3-hydroxybutyrate triolide, and the solvent is tetrahydrofuran.
A method as claimed in Claim 1 wherein the 1,3-butandiol is racemic and the product is at least 95% mono-adduct with R being H.
A method as claimed in Claim 1 wherein the alcohol is (R)-1,3-butandiol.
A method as claimed in Claim 5 wherein the solvent is a mixture of tetrahydrofuran and toluene in ratio in the range 10:90 to 90:10.
A method as claimed in Claim 6 wherein the product is a mixture of mono and di-adducts of H(OCH(CH₃)CH₂C(O))₃- with (R)-1,3-butanediol in ratio of 10:90 to 90:10.
A method as claimed in Claim 6 wherein the products are purified by column chromatography using a methanol: chloroform mixture.
A method as claimed in Claim 8 wherein the mixture is between 1 and 5% methanol and 99 to 95% chloroform v/v.
A process for producing a nutraceutical or pharmaceutical composition comprising a method as claimed in any one of Claims 1 to 9.